JPH0618412A - Scanning refractive index microscope - Google Patents

Abstract

PURPOSE:To provide a scanning microscope that is able to eliminate any effect of transmission factor and reflection factor of a sample and to visualize the refractive index distribution aright. CONSTITUTION:A transmitted light 11' out of a sample 23 is imaged to a point image Q by a condenser lens 27, and the quantity of this transmitted light 11' is detected by a photo detector 29 via a pinhole plate 28 set up in and around this imaged position. In addition, pinhole plate 28 and photodetector 29 are shifted in an optical axial direction (z direction) by a piezoelectric element 51, while a position of the pinhole plate 28 at a time when light quantity data at each picture elect varying with this shifting takes the maximum value is sought at each picture element by a computer 55. Then, a sample refractive index at each picture element is operated on the basis of a specified formula from a position of the pinhole plate 28 by this computer 55.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning microscope, and more particularly to a scanning refractive index microscope for visualizing the refractive index distribution of a sample.

[0002]

2. Description of the Related Art Conventionally, illumination light is converged on a minute light spot, and this light spot is two-dimensionally scanned on a sample, and at that time, light transmitted through the sample or light reflected there, and further a sample The fluorescence emitted from is detected by a photodetector,
Optical scanning microscopes are known which are adapted to obtain an electrical signal carrying an enlarged image of the sample.

This optical scanning microscope is basically a sample stage on which a sample is placed, a light source for emitting illumination light, and a light-sending optical system for forming an image of this illumination light as a minute light spot on the sample. A light-receiving optical system that collects the light flux (transmitted light, reflected light, or fluorescence) from the sample to form a point image, a photodetector that detects the point image, and the light point on the sample. It is composed of a scanning means for two-dimensionally scanning. An example of this optical scanning microscope is shown in Japanese Patent Laid-Open No. 62-217218.

On the other hand, conventionally, a phase contrast microscope has been provided as one of the microscopes for observing the refractive index distribution of a transparent object. This phase-contrast microscope basically has a phase plate such as a λ / 4 plate and a ring diaphragm that are arranged on opposite sides of the sample, and the illumination light diffracted by the sample and transmitted through the sample or there. In this method, only one of the reflected non-diffracted illumination light is given a phase delay and then interfered, thereby converting the refractive index distribution of the sample into light and dark. Also,
As disclosed in Japanese Patent Laid-Open No. 4-156507, a scanning phase contrast microscope has already been proposed.

[0005]

However, in the phase contrast microscope as described above, the amount of transmitted light or reflected light from the sample changes depending on the transmittance or reflectance of the sample in addition to the refractive index of the sample. Therefore, the pure refractive index distribution of the sample may not be correctly observed.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a scanning refractive index microscope capable of accurately visualizing the refractive index distribution of a sample.

[0007]

A scanning type refractive index microscope according to the present invention comprises a sample stage, a light source, a light-sending optical system, a light spot two-dimensional scanning means, and a transmitted light from a sample as described above. In a scanning microscope including a light-receiving optical system for forming a point image and a photodetector for detecting the point image, an aperture arranged near the point image forming position and the output of the photodetector. The signal is divided into pixels to obtain a light quantity signal for each pixel on the sample, a means for moving the diaphragm in the optical axis direction, and a light quantity signal for each pixel that changes with the movement of the diaphragm is maximum. An aperture position detecting means for obtaining the aperture position when taking a value for each pixel and an arithmetic means for obtaining the sample refractive index for each pixel based on the aperture position obtained by the aperture position detecting means are provided. It is a thing.

[0008]

ACTION AND EFFECT OF THE INVENTION As an example, consider the system shown in FIG. In this system, the illumination light 11 is focused on the light point P in the sample 23 by the action of the objective lens 17 which constitutes a part of the light transmission optical system. The transmitted light 11 ′ that has passed through the sample 23 is formed into a point image Q by the objective lens 19 and the condenser lens 27 that form the light receiving optical system. Then, the light quantity of this point image Q is detected by the photodetector 29 via the pinhole plate 28 as a diaphragm.

Here, when the refractive index of the sample 23 in the portion irradiated by the light spot P is n ₀ , the transmitted light 11 'is emitted from the objective lens 19 in a parallel light state as shown in FIG. And
In this case, when the pinhole plate 28 is at the position F ₃ ′ in the figure, that is, at the rear focal point of the condenser lens 27, the amount of light detected by the photodetector 29 is maximum. Next, when the refractive index of the sample 23 in the portion of the light point P is n ₁ which is different from n ₀ , it is assumed that the optical path of the transmitted light 11 ′ is changed as shown in FIG. In this case, since the point image Q is formed at a position z ₃ ′ away from the rear focal point F ₃ ′, when the pinhole plate 28 is located at this position, the amount of light detected by the photodetector 29 is the maximum. (More preferably, the photodetector 29 is also moved integrally with the pinhole plate 28).
Since the above-mentioned distance z ₃ 'changes according to the relationship determined by n ₀ and n ₁ , if the refractive index n ₀ is investigated beforehand using a reference sample, it is based on this amount of z ₃ ' The refractive index n ₁ will be known.

The above relationship will be described below. Figure 5
Each condition of the system is shown in FIG. That is, the refractive indices of the media on the front side and the rear side of the objective lens 19 are n ₂ and
n ₂ ′, similarly, the refractive indices of the media on the front side and the rear side of the condenser lens 27 are n ₃ (= n ₂ ′), n ₃ ′, and the objective lens 19 respectively.
And the focal lengths of the condenser lens 27 are f ₂ , f ₃ , respectively, and the front and rear focal points of the objective lens 19 are F ₂ , respectively.
F ₂ 'and the front and rear focal points of the condenser lens 27 are respectively F ₃
And F ₃ ′, the distance between the front focus F ₂ of the objective lens 19 and the light spot P is z ₂ , and the distance between the rear focus F ₃ ′ of the condenser lens 27 and the point image Q is z _{3 as described above.} 'And focus F
The distance between the ₂ 'and F ₃ and L. Considering the image forming position by the objective lens 19, the distance between this image forming position and the rear focus F ₂ ′ of the objective lens 19 is z ₂ ′, and this image forming position and the front focus F ₃ of the condenser lens 27 are Given that the distance is z ₃ , from Newton's formula,

[0011]

[Equation 1]

[0012] If the thickness of sample 23 is d

[0013]

[Equation 2]

Therefore, by substituting this into the above equation (1),
In particular, L = 0 and n ₂ = n for use in air
If _{2 '= n 3 = n 3} ' = 1 and,

[0015]

[Equation 3]

[0016] From the above, if the sample thickness d and the focal lengths f ₂ and f ₃ are known, the sample refractive index n ₁ can be known from the distance z ₃ ′ based on the equation (2).

It is convenient to set L = 0 as described above, but it is not necessary to do so. However, when L ≠ 0, the relationship between z ₂ and z ₃ 'shown by the above equation (1) is approximately as shown in FIG. 7, and if the change range of z ₂ is, for example, the range of W in the same figure. In that case, z ₂ and z ₃ ′ do not uniquely correspond to each other. This means that z ₃ ′ and the sample refractive index n ₁ do not uniquely correspond to each other after all.
In order to avoid such a situation, when the assumed maximum absolute value of z ₂ is Δz,

[0018]

[Equation 4]

It suffices to set L so as to satisfy the above condition.

As described above, the scanning refractive index microscope of the present invention visualizes the sample refractive index based on the change in the image forming position of the transmitted light due to the difference in the refractive index of the sample, not the light amount of the transmitted light itself. With such a configuration, it is possible to obtain pure refractive index information of the sample without being affected by the light transmittance and reflectance of the sample.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below based on the embodiments shown in the drawings. FIG. 1 shows a confocal scanning refractive index microscope according to an embodiment of the present invention, and FIG. 2 is a II-II line arrow view thereof. The scanning refractive index microscope of this embodiment is formed so as to be usable also as a normal scanning microscope for capturing a magnified image of a sample. First, the configuration of a normal scanning microscope will be described.

As shown in the drawing, a laser beam 11 as illumination light is emitted from a monochromatic laser 10 such as an Ar laser.
Is ejected. The illumination light 11 is collimated by a collimating optical system 12 including lenses 12a and 12b, travels in the direction of arrow X, that is, in the horizontal direction, and is incident on a mirror 16 fixed to a moving table 15.

The mirror 16 is fixed on the movable table 15 at an angle of 45 ° with respect to the X direction.
Is emitted downward from the mirror 16 (that is, in the Z direction). The movable table 15 holds an objective lens (condenser lens) 17 that constitutes a light-sending optical system together with the collimating optical system 12. Further, the movable table 15 holds an objective lens 19 which constitutes a part of the light receiving optical system. The two lenses 17 and 19 are fixed so that their optical axes coincide with each other. A sample table 22 which is a separate body from the movable table 15 is arranged between the lenses 17 and 19. And moving table 15
The mirror 20 is fixed to the mirror 20 in a state of being inclined by 45 ° with respect to the lens optical axis. The mirror 20 reflects the transmitted light 11 ′ transmitted through the sample 23 mounted on the sample table 22 in the X direction (direction opposite to the above).

The illumination light 11 in the parallel light state is condensed by the objective lens 17 and converges on a minute light spot (beam spot) P on the sample 23 (surface or inside) mounted on the sample table 22. The light flux of the transmitted light 11 'transmitted through the sample 23 is the objective lens.
The light is made into parallel light or a state close to it by 19 and a part of the light passes through the mirror 20 and is then condensed by the condenser lens 24 to form a point image Q1. The light quantity of the point image Q1 is detected by a photodetector 25 such as a photomultiplier tube through the confocal pinhole plate 26, and a transmitted light detection signal S1 indicating this light quantity is output from the photodetector 25.

Next, the two-dimensional scanning of the illumination light spot P will be described. A main scanning laminated piezoelectric element 37 is interposed between the movable table 15 and the gantry 36. The laminated piezo element 37 is driven by receiving driving power from the piezo element driving circuit 38, and reciprocates the moving table 15 at high speed in the X direction orthogonal to the optical axes of the lenses 17 and 19. On the other hand, laminated piezo elements 40 and 41 are interposed between the sample table 22 and the gantry 36. The laminated piezo element 40 is driven by receiving drive power from the piezo element drive circuit 42, and reciprocates the sample stage 22 at high speed in the Y direction. As a result, the sample table 22 is moved relative to the movable table 15, and the light spot P sub-scans the sample 23 in the Y direction orthogonal to the main scanning direction X. As described above, the illumination light spot P is the sample 23.
By scanning the top two-dimensionally, a signal S1 carrying a two-dimensional magnified image of the sample 23 is obtained.

If this signal S1 is sampled and quantized based on a predetermined pixel clock and then input to a display device such as a CRT display device or an image reproducing device 65 such as an optical scanning recording device, the signal S1 is obtained. It becomes possible to reproduce and observe the microscope image carried by.

The sub-scanning laminated piezoelectric element is provided at the upper end.
40 is fixed, and the lower end is attached to the pedestal 36 via the coarse movement stage 44.
The laminated piezo element 40, which is driven by receiving driving power from 43 and holds the sample table 22, is perpendicular to the main and sub scanning directions X and Y.
Direction (the optical axis direction of the lenses 17 and 19). This enables focusing adjustment.

The piezo element drive circuits 38 and 42 include
A synchronization signal is input from the control circuit 45, whereby the main scanning and the sub scanning of the light spot P are synchronized. Further, the coarse movement stage 44 can be largely moved in the Y direction manually or by using a driving means. By moving the sample table 22 in this way, the sample 23 can be easily replaced.

Next, the structure of this scanning microscope as a refractive index microscope will be described. A part of the transmitted light 11 'is reflected by the mirror 20 and emitted to the outside of the moving table 15. This transmitted light 1
1'is condensed by a condenser lens 27 which constitutes a light receiving optical system together with the objective lens 19 and forms a point image Q. The light quantity of the point image Q is detected by a photodetector 29 such as a photomultiplier tube via the confocal pinhole plate 28, and a light quantity signal S indicating this light quantity is output from the photodetector 29.

The pinhole plate 28 and the photodetector 29 are integrally held by the moving stage 50. This moving stage 50 uses a piezo element 51 to move in the z direction, that is, a condenser lens.
It is movable in the 27 optical axis directions. The displacement of the moving stage 50, that is, the moving position of the pinhole plate 28 is detected by a displacement gauge 52, and its displacement output Sz is amplified by an amplifier 53 and then input to a feedback control circuit 54. This displacement is due to the rear focus F ₃ 'of the condenser lens 27.
It is defined as the distance between the pinhole plate 28 (see FIG. 5) and the pinhole plate 28.

The feedback control circuit 54 outputs a drive signal Mp according to the displacement designation signal Mz input from the computer 55. The drive signal Mp is amplified by the amplifier 56 and then input to the piezo element 51.
The piezo element 51 is driven based on p. The feedback control circuit 54 determines that the actual displacement of the moving stage 50 indicated by the displacement output Sz input thereto is the displacement designation signal Mz.
The drive signal Mp is controlled so as to match the target displacement indicated by.

Next, the structure of the sample refractive index image by the computer 55 will be described. The light amount signal S output from the photodetector 29 is amplified by the amplifier 57, sampled and quantized by the A / D converter, and then converted into digital light amount data I.
Is converted to. The light amount data I is input to the computer 55. In the present embodiment, as an example, an image having 512 pixels in the main scanning direction and 480 pixels in the sub scanning direction is formed.
That is, when the light amount data for the m-th pixel in the main scanning direction and the n-th pixel in the sub-scanning direction is expressed as I (m, n),
The computer 55 has I (1,1), I (2,1), I
(3,1) ... I (512,1), I (1,2), I
(2, 2) ... Light quantity data is input in the order of I (512, 480).

The computer 55 has a line buffer memory 60 for temporarily storing 512 light amount data I for one main scanning line, and a first memory 61 for storing one image, that is, 512 × 480 light amount data I. , 512 for one image
Second memory for storing x480 movement stage displacement data
62 and.

Next, formation of sample refractive index data by the computer 55 will be described with reference to FIG. 3 showing a basic flow of the processing. When this process starts, the computer 55 first moves to the moving stage in step P1.
Position 50. The moving stage 50 is moved from the initial position z _p in the z direction to the final position z _{e with} a predetermined small pitch shift, and is initially positioned at the initial position z _p . This computer 55
Specifically, the positioning operation by is the output of the displacement designation signal Mz described above.

Next, in step P2, the computer 55 starts main scanning and sub scanning of the illumination light spot P. This scanning start command is specifically an operation of inputting a piezo element drive start signal to the control circuit 45. Then computer
In step P3, 55 is 512 for the first main scanning line.
Each light quantity data Inew for the pixel is stored. Next, in step P4, the light amount data Iold and the new light amount data Inew for the 512 pixels of the first main scanning line stored in the first memory 61 are divided into the first, second and third values. ... The data of the common pixel is compared for each data in the order of the 512th pixel. Here, the first memory 61 is initially
As 512 × 480 light quantity data Iold, data indicating a light quantity of zero is stored.
Iold <Inew for all two pixels,
The process proceeds to step P5.

In step P5, the light amount data Iold for each pixel stored in the first memory 61 is replaced with new data Inew for the pixel larger than that, and at the same time, the computer
The position z of the moving stage 50 designated by 55 is the second memory
It is stored in the address of 62 for the pixel.

Next, at step P6, it is judged if the number m of times the process has flown to this step P6 has reached 512. If not, the process flow goes to step P6.
Return to 4. As described above, all 512 new light amount data Inew relating to the first main scanning line are stored in the first memory 61.
Memorized in.

If it is determined in step P6 that m = 512, the flow of processing moves to step P7. This step P
In step 7, it is judged whether or not the number n of times the process has flown to step P7 has reached 480. If not, the flow of processing returns to step P3. As described above, regarding the 1st main scanning line to the 480th main scanning line
All 512 × 480 new light intensity data Inew are stored in the first memory 61.

If n = 480 is determined in step P7, it is then determined in step P8 whether or not the position z of the moving stage 50 has reached the final position z _e. Position of stage 50 z
Is changed to the final position z _e by a predetermined pitch, and the flow of processing returns to step P1. Thus, every time the position z of the moving stage 50 is changed, the above-mentioned step P1
The processes from to P7 are performed.

After the position z of the moving stage 50 has been changed once in step 9, that is, for the second and subsequent images, in step 4 Iold <Inew
Of course, it may be determined that it is not. In that case,
The process flow moves to step P6. So in this case,
The light amount data Iold stored in the first memory 61 is left as it is. The above process is performed in step P8.
= When done until it is determined that z _e, the second memory 62, so that the z position when the maximum light quantity data I is obtained for each pixel of 512 × 480 pieces is stored.

The computer 55, when it is judged z = z _e in step P8, in step P10, the second
The sample refractive index n ₁ for each pixel is calculated based on the z position data stored in the memory 62. That is, the maximum light amount data I is obtained when the pinhole plate 28 is located exactly at the image forming position of the point image Q of FIG. 5, and the z position at this time is z ₃ 'of FIG. The position. Therefore, the computer 55 calculates the sample refractive index n ₁ from the value of z ₃ ′ based on the above-mentioned equation (2). Here, using a reference sample whose refractive index n ₀ is known in advance, its thickness d is measured, and the focal lengths f ₂ and f ₃ are measured,

[0042]

[Equation 5]

From the equation (2),

[0044]

[Equation 6]

As a result, the refractive index n ₁ of the sample 23 is obtained.

Since the refractive indices n ₀ and k are constants, the relationship between z ₃ 'and n _{1 in} the above equation is approximately as shown in FIG. As is clear from this figure, the refractive index n ₀ can be uniquely obtained according to the value of z ₃ ′.

The computer 55 is thus 512
The refractive index n ₁ is calculated for each of the × 480 pixels, and the refractive index n ₁ is calculated.
_The image data Sn obtained by converting the value of ₁ into the density value is output. By inputting this image data Sn into an image reproducing device 65 such as a CRT display device or an optical scanning recording device, it is possible to reproduce an enlarged image of the sample 23 in which the refractive index distribution shows a density change.

In this embodiment, the illumination light 11 is used as the moving table.
Since the light travels in the moving direction of 15, i.e., the main scanning direction X, and enters the mirror 16, the illumination light 11 in the moving table 15 is always in a parallel light state regardless of the position of the moving table 15 in the X direction. The beam center is kept in alignment with the optical axes of lenses 17, 19 and 24. Further, since the transmitted light 11 'is also emitted from the mirror 20 in the same direction as the main scanning direction X, the moving table 15'
The beam center of the transmitted light 11 ′ is kept aligned with the optical axis of the lens 27 regardless of the position in the X direction.

Depending on the scanning method of the illumination light light point P, when the transmitted light 11 'emitted from the objective lens 19 is not in the parallel light state, as shown by the broken line in FIG. The point image Q may move. In such a case, instead of the pinhole plate 28 as a diaphragm placed in front of the photodetector 29, a slit plate having a slit extending along the point image moving direction may be used.

[Brief description of drawings]

FIG. 1 is a schematic side view showing a scanning refractive index microscope according to an embodiment of the present invention.

FIG. 2 is a view taken along the line II-II in FIG.

FIG. 3 is a flowchart showing a flow of processing for forming sample refractive index data in the scanning refractive index microscope.

FIG. 4 is a graph showing an outline of a relationship between a diaphragm position and a sample refractive index in the scanning refractive index microscope.

FIG. 5 is a side view showing a main part of an optical system of the scanning refractive index microscope.

FIG. 6 is a schematic diagram for showing optical conditions in the optical system of FIG.

FIG. 7 is a graph showing the outline of the relationship between the distance between the front focus and the illumination light spot of the objective lens and the distance between the rear focus and the point image of the condenser lens in the scanning refractive index microscope of the present invention.

[Explanation of symbols]

 10 Monochromatic laser 11 Illumination light 11 'Transmitted light 12 Collimating optical system 15 Moving stage 20 Mirror 17, 19 Objective lens 22 Sample stage 23 Sample 24, 27 Condensing lens 25, 29 Photodetector 26, 28 Confocal pinhole plate ( Aperture) 36 Stand 37, 40, 41 Multilayer piezo element 38, 42, 43 Piezo element drive circuit 45 Control circuit 50 Moving stage 51 Piezo element 52 Displacement meter 54 Feedback control circuit 55 Computer 58 A / D converter 60 Line buffer memory 61 First memory 62 Second memory 65 Image playback device

Claims (1)

[Claims] 1. A sample table on which a sample is placed, a light source that emits illumination light, a light transmission optical system that forms an image of the illumination light as a minute light spot on the sample, and a transmitted light from the sample. A light receiving optical system for condensing and forming an image in a point image, a diaphragm arranged near the image forming position of the point image, a photodetector for detecting the transmitted light passing through the diaphragm, and the light spot A means for two-dimensionally scanning on the sample; a means for dividing the output signal of the photodetector into pixels to obtain a light quantity signal for each pixel on the sample; and a means for moving the diaphragm in the optical axis direction. Based on the aperture position detection means for obtaining for each pixel, the aperture position when the light amount signal for each pixel, which changes with the movement of the aperture, takes the maximum value, and the aperture position obtained by this aperture position detection means. A calculation means for calculating the sample refractive index for each pixel. Type refractive index microscope.